Pharmaceutical aerosol composition

ABSTRACT

An aerosol formulation comprising a biodegradable microsphere comprising a non-living reagent, such as a sub-unit vaccine, that produces a protective immune response in a mammal to whom it is administered. Nebulizers and inhalers containing such formulations are also described and claimed.

The present invention relates to pharmaceutical compositions, and inparticular to compositions comprising immunogens, used in theprophylactic and therapeutic treatment of infections.

The option to self administer vaccines by inhalation, for example usinga nebulizer or inhaler such as a dry powder inhaler, would beadvantageous from a logistical standpoint and may be particularlyeffective for protecting individuals from pathogens that affect orutilise the respiratory tract as a portal of entry into the body.

U.S. Pat. No. 6,428,771 describes a method for controlled drug deliveryto the pulmonary system using microparticles incorporating the drug.Particles are described as having a diameter of from 0.5 and 10 μm. Itis suggested that the drug may in fact comprise an antigen intended toelicit an a protective immune response. However this is notdemonstrated.

Furthermore, administration of in particular non-living vaccines, suchas sub-unit vaccines, has not yet been found to give effectiveprotection using this mode of administration (see for example C. W.Purdy et al., Current Microbiology, (1998), 37, p5).

The applicants have found that biodegradable microspheres, containingantigen, can engender immunological responses following delivery toexperimental animals in the form of an aerosol, provided themicrospheres are of a type which are delivered most efficiently to thelung.

According to the present invention there is provided an aerosolformulation comprising a biodegradable microsphere of average diameterof from 0.5 to 5 μm and comprising a non-living reagent that produces aprotective immune response in a host mammal to whom it is administered.

As used herein, the term “non-living reagent” refers to immunogens suchas polypeptides or proteins, which are derived for example from apathogen such as a bacteria, virus or fungi. It also refers toinactivated microorganisms such as heat or chemically killed bacteriaand/or viruses.

The term “aerosol” refers to a formulation that is deliverable in theform of a dispersion of a solid and/or liquid in a gas. These may beprepared from suspensions of the formulation in a liquid such as water,using a device such as a nebulizer, or from dry powders using a drypowder inhaler. In the case of the nebulized aerosol, the dispersioncomprises essentially wet microspheres in air.

The term “average diameter” as used herein, refers to the mean massaerodynamic diameter of the microspheres. Mean mass aerodynamic diameteris a measurement of particle size in an aerosol, which is the mostrelevant measurement when trying to predict if particles are respirable.

These formulations are effective in the administration of reagents,which are capable of generating a protective immune response in ananimal, particularly a mammal, to which it is administered. Examples ofsuch agents include antigenic polypeptides as well as nucleic acidsequences which may encode these polypeptides and which are known as“DNA” vaccines.

Suitable polypeptides are sub-unit vaccines and others, such asdiptheria toxoid, tetanus toxoid, Botulinun toxin FHc and Bacillusanthracis protective antigen (PA).

As used herein the expression “polypeptide” encompasses proteins orepitopic fragments thereof.

Suitable polypeptides are sub-unit vaccines.

In a preferred embodiment, the formulation of the invention comprises abiologically active agent which is capable of generating a protectiveimmune response against Yersinia pestis. The agent is suitably asub-unit vaccine, for example V antigen of Y. pestis or animmunologically active fragment thereof or a variant of these, or the F1antigen of Y. pestis or an immunologically active fragment thereof or avariant of these, or a combination of these. In particular as describedin WO 96/28551, preferred vaccine comprises a combination of the F1 andV antigens.

As used herein, the term “fragment” refers to a portion of the basicsequence that includes at least one antigenic determinant. These may bedeletion mutants. One or more epitopic region of the sequence may bejoined together.

The expression “variant” refers to sequences of nucleic acids thatdiffer from the base sequence from which they are derived in that one ormore amino acids within the sequence are substituted for other aminoacids. Amino acid substitutions may be regarded as “conservative” wherean amino acid is replaced with a different amino acid with broadlysimilar properties. Non-conservative substitutions are where amino acidsare replaced with amino acids of a different type. Broadly speaking,fewer non-conservative substitutions will be possible without alteringthe biological activity of the polypeptide. Suitably variants will be atleast 60% identical, preferably at least 75% identical, and morepreferably at least 90% identical to the base sequence. Identity in thiscase can be determined using available algorithms such as the widelyused BLAST program.

The applicants have found that nebulization of PLA microspheresgenerates a respirable ‘plume’ of aerosolised particles, and thisapproach can be used to deliver immunogens to the respiratory tracts ofexperimental animals. Similar plumes could be produced using other formsof inhaler such as dry powder inhalers.

Microspheres used are suitably small enough to allow them to beadministered to the deep lung using a conventional nebulizer or inhaler.For this purpose, microspheres will be less that 5 μm average diameter,preferably less than 3 μm average diameter, for instance from 0.5-3 μm,or more preferably from 1-3 μm and most preferably with an averagediameter of between 1 and 1.5 μm.

Suitably 0% of microspheres have an aerodynamic diameter above 10 μm.More suitably, 0% of microspheres have an aerodynamic diameter above 9μm, and preferably 0% of microspheres have an aerodynamic diameter above6 μm.

Suitably, at least 90%, and preferably at least 95% of the microspheresin the formulation have an aerodynamic diameter of less than 5 μm,preferably with at least 80% of particles having a mean mass aerodynamicdiameter of less than 3 μm.

By using microspheres of this size, efficient delivery of reagent intothe deep lung is achieved. This is important in the delivery of reagentsof this type as it is essential to achieve the highest concentrations ofreagent, which can feasibly and safely be delivered in order to achievethe protective immune response.

Microspheres are suitably biodegradable and are produced from polymericmaterial. The polymeric material is suitably a biogdegradable polymerother than a lipid, and in particular a biodegradable polyester. Aparticularly suitable polymer for use in the preparation ofmicrocapsules is Poly-lactide (PL) although other polymers such aspoly(lactide-co-glycolide) PLGA may also be employed.

The microspheres may optionally further comprise agents which stabiliseemulsions such as polyvinylalcohol (PVA), dipalmitoylphophatidylcholine(DPPC), or methyl cellulose, and preferably polyvinylalcohol.

Suitably the non-living reagent is encapsulated within the microspheres(microcapsules). This again ensures the a high dose of the reagent isdelivered to the lung which is important if a protective immune responseis to be generated.

Microcapsules are suitably prepared using conventional methods such asthe double emulsion/solvent evaporation method, as described for exampleby Beck et al., 1979, Fertility and Sterility, 31:545-551.

The encapsulation is suitably achieved using a double emulsion solventevaporation method, in which a first emulsion is formed with thenon-living reagent, and the structural polymer, mixing this with anaqueous phase (suitably without structural polymer) to form a secondaryemulsion, evaporating solvent and isolating small microspheres. Inparticular, the pharmaceutically active ingredient is dissolved orsuspended in an aqueous solution, which optionally includes anemulsifier such as PVA. The emulsifier, where present is suitablyincluded at low concentrations for example of less than 5% w/v. Thissolution or suspension in then mixed with a solution of the highmolecular weight structural polymer in an organic solvent such asdichloromethane. A primary emulsion is then formed, in particular bysonication of the mixture. The primary emulsion in then added to asecondary aqueous phase, which preferably includes an emulsifier withvigorous stirring. Solvent is then preferably evaporated, convenientlyat room temperature.

Microspheres can then be recovered, for example by centrifugationfollowed by lyophilisation.

The formulations of the invention may comprise microspheres per se whichare optionally preserved, for example by lyophilisation, or themicrospheres may be combined with a pharmaceutically acceptable carrieror excipient. Examples of suitable carriers include solid carriers as isunderstood in the art for use in nebulizers.

In a particularly preferred embodiment, the formulation furthercomprises the non-living reagent in free form. The ratio of the amountsof the free reagent to the reagent associated with the microspheres usedin the composition may vary depending upon the particular agents beingemployed. Suitably the ratio of the free reagent to the reagentcontained in the microspheres is in the range of from 1:20 to 2:1 andpreferably at about 1:10.

The formulation of the invention may further comprise an adjuvant inorder to enhance the immune response to the biologically active materialadministered. Suitable adjuvants include pharmaceutically acceptableadjuvants such as Freund's incomplete adjuvant, alhydrogel, aluminiumcompounds and, preferably adjuvants which are known to up-regulatemucosal responses such as CTB, the non-toxic pentameric B subunit ofcholera toxin (CT) or mutant heat-labile toxin (mLT) of E. coli. Theymay also include immunomodulators such as cytokines and CpG motifs.

Other adjuvant types are described in International Patent ApplicationNos. WO00/56282, WO00/56362 and WO00/56361.

Suitably the formulations are in unit dosage form. This will varydepending upon the nature of the active agent being employed, the natureof the patient, the condition being treated and other clinical factors.In general however, the formulations of the invention will compriseapproximately 0.5 to 10 w/w of non-living reagent.

Exposed animals, in this case, mice, respond with a humoral response. Ithas also been found that experimental animals can be protected by thistreatment from a lethal challenge with a pathogen such as the plaguecausing bacteria (Yersinia. pestis) by exposure to aerosolisedmicrospheres containing recombinant V antigen. The applicants aretherefore the first to demonstrate the successful aerogenic immunisationusing non-living vaccines.

Dosages of the formulations of the invention will depend upon variousfactors such as the the nature of the patient, the antigen used etc. andwill be determined according to known clinical practice.

It has been found that in a particularly preferred embodiment, eachadministration of microsphere preparation to a mouse contains from 1-100μg, suitably from 30-50 μg and most preferably about 40 μg of each ofsaid antigens. Preferably the dosage to humans and mammals would be ofthe same order in terms of mg/Kg.

According to a further aspect of the invention, there is provided anebulizer or inhaler comprising a formulation as described above.

Dry powder inhalers may be particularly useful in the context of theinvention as dry vaccine formulations, which would be used therein, arestable at ambient temperatures.

In yet a further aspect, the invention provides the use of microspherescomprising a non-living reagent that produces a protective immuneresponse in a mammal to whom it is administered, in the preparation of avaccine for administration as an aerosol.

Further according to the invention there is provided a method ofproducing a protective immune response in a mammal in need thereof, saidmethod comprising administering to the lung of said mammal, a protectiveamount of an aerosol formulation as described above.

The invention will now be particularly described by way of example withreference to the accompanying diagrammatic drawings in which:

FIG. 1 is a micrograph showing the morphology of microspheres prior to(A) and after (B) nebulization;

FIG. 2 is a graph showing serum anti-V IgG endpoint titre in 6 BALB/cmice exposed to aerosolised microspheres containing recombinant Yersiniapestis V antigen; and

FIG. 3 illustrates the survival of mice, previously exposed toaerosolised microspheres containing rV antigen, after subcutaneousinjection of 6.5 MLDs Y. pestis;

FIG. 4 is a fluorescence micrograph of lung taken 24 hours followingexposure of mice to aerosolised microspheres loaded with FITC-BSA; and

FIG. 5 is a fluorescence micrograph of lung lymph node taken 24 hoursfollowing exposure of mice to aerosolised microspheres loaded withFITC-BSA.

EXAMPLE 1

Poly-lactide (resomer L210) microspheres containing either BSA orrecombinant V antigen from Y. pestis were fabricated using a modifieddouble-emulsion solvent evaporation process. PLA, sold under the tradename Resomer L210, is a linear crystalline homopolymer with an inherentviscosity of approximately 3.6.

The polymer was used at a concentration of 1.38% w/v in dichloromethane(10 ml). An aqueous solution (0.5 ml) containing the antigen of interest(about 4 mg) was then added and the mixture stirred at high speed togenerate an emulsion. This emulsion was then added to a second aqueousphase and mixed together at high speed.

The solvent was then evaporated to leave an aqueous suspension ofantigen-loaded microspheres.

Particles were aerosolised using a Sidestream® nebulizer. An aerosolparticle sizer was used to analyse size characteristics. Samples werecollected using a three stage liquid impinger and analysed usingscanning electron microscopy, SDS PAGE and western blotting procedures.

6 female BALB/c mice were exposed to a stream of aerosolisedmicrospheres in a head only exposure line. 77 mg of rV loadedmicrospheres were suspended in 17 ml of free V (at 0.4 mg ml⁻¹ indistilled water). Mice were exposed to the aerosolised microspheres forthree ten minute runs, during which time approximately 3 ml of particlesuspension was nebulized each run. The was repeated on days 0, 21 and107 of the experiment and sera analysed for the presence of anti-V IgGusing an indirect ELISA. In order to assess the extent of protectionafforded by inhalation of the V loaded microspheres, mice were injectedsubcutaneously with 6.3MLDs Y. pestis (GB strain) on day 136 of theexperiment.

Results and Discussion

Microspheres had a loading of 3.8% w/w (BSA) and 3.3% w/w (rV).Following aerosolisation the BSA loaded particles had a mass medianaerodynamic diameter of 1.3±1.4 μm, with 93% of the particles under 3μm. Following nebulization, particles retained theirmorphology/topography (FIG. 1) and contained antigenic material asdetected by Western Blotting.

Although there was some inter-animal variation in the serum antibodyresponse to aerosolised Y. pestis rV antigen, all 6 mice seroconvertedafter three immunising doses (FIG. 2). Two of the six mice respondedwith antibody titres that were of significant magnitude to conferprotection from injected challenge with plague causing bacteria (FIG.3).

EXAMPLE 2

Delivery of Microencapsulated Antigen to the Lung and Lung Lymph Node byAerosolisation

Poly-lactide (resomer L210, Alfa chemicals UK) microspheres, containingFITC-BSA were fabricated using a modified double-emulsion solventevaporation process. Particles were aerosolised using a Sidestream®nebulizer (Profile, UK). Female BALB/c mice were exposed to theaerosolised microspheres in a head only exposure chamber. 24 hoursfollowing exposure mice were killed and their lungs and lung's lymphnodes were extracted. Frozen sections were obtained from the extractedtissues using a cryostat. Frozen sections were examined for the presenceof FITC-BSA loaded microspheres using a fluorescence microscope and theresults for the lung and lung lymph nodes are shown in FIGS. 4 and 5respectively.

The visualisation of punctate fluorescent material in the sectionsindicated the presnce of FITC-BSA loaded microspheres in the lung andlymph nodes. These data support the tenet that microspheres can reachenter the lower respiratory tract following nebulization. Furthermore,these data indicate that microspheres may be translocated from the lungsto the draining lymph nodes, following nebulization.

1. An aerosol formulation comprising a biodegradable microsphere ofaverage diameter of from 0.5 to 5 μm comprising a non-living reagentthat produces a protective immune response in a mammal to whom it isadministered.
 2. The formulation of claim 1 wherein the said non-livingreagent is antigenic polypeptide or a nucleic acid sequences which mayencode such a polypeptide.
 3. The formulation of claim 2 wherein thesaid non-living reagent is a sub-unit vaccine.
 4. The formulation ofclaim 1 wherein the said non-living reagent is diptheria toxoid, tetanustoxoid, Botulinum toxin FHc, Bacillus anthracis protective antigen (PA)or a polypeptide which is capable of generating a protective immuneresponse against Yersinia pestis.
 5. The formulation of claim 4 whereinthe non-living reagent is the V antigen of Y. pestis or animmunologically active fragment thereof or a variant of these, or the F1antigen of Y. pestis or an immunologically active fragment thereof or avariant of these, or a combination of these.
 6. The formulation of claim1 wherein the microspheres have an average diameter of less than 3 μm.7. The formulation of claim 6 wherein the microcapsules have an averagediameter of between 1 and 1.5 μm.
 8. The formulation of claim 1 whereinthe microspheres comprise a biodegradable polyester.
 9. The formulationof claim 8 wherein the polyester comprises Poly-lactide (PL).
 10. Theformulation of claim 1 wherein the microcapsules are lyophilised. 11.The formulation of claim 1 wherein the non-living reagent isencapsulated within the microspheres.
 12. The formulation of claim 1which further comprises the non-living reagent in free form.
 13. Theformulation of claim 12 wherein the ratio of the amounts of the freereagent to the reagent associated with the microspheres is in the rangeof from 1:20 to 2:1.
 14. The formulation of claim 1 in unit dosage form.15. A nebulizer or inhaler comprising a formulation of claim
 1. 16.(canceled)
 17. A method of producing a protective immune response in amammal in need thereof, said method comprising administering to the lungof said mammal, a protective amount of an aerosol formulation of claim1.